Many common substances, such as blood, soil and tap water, are actually mixtures of different materials combined in varying proportions. For example, a blood sample is a complex mixture of proteins, cells and other materials combined in different proportions and in different concentrations throughout the volume of the sample. Often a major task during the analysis of these substances is separating the components out of the mixture so that they can be studied in isolation.
One known technique for separating the components of a mixture binds magnetic beads to the particles of interest and draws the particles of interest out of the mixture through the application of a magnetic field. Typically these magnetic assays include paramagnetic beads and an agent that binds with particles of the desired component group. The agent is selected for its specificity to the particle of interest. For example, to select the positively charged particles dispersed in a mixture, a negatively charged agent can be selected. In another example, binding agents can be selected that have antigen specificity for a protein particle in the mixture. Commonly, the agent is bound to the surface of the magnetic bead so that when the agent reacts with selected particles it binds the paramagnetic bead to the particle. The particles bound to the paramagnetic beads can be isolated out of the mixture by applying a magnetic field to the mixture to remove the bead, and the bound particle, from the mixture.
In one particular magnetic separation assay, the MACS Cell Sorter, High Gradient Magnetic Cell Separation with MACS, Miltenyi et al., Ctyometry 11:23 1-238 (1990), a magnetic filter is formed that collects cells bound to superparamagnetic particles. The cells are contained in a fluid mixture placed in a sterile container. Magnetic beads are coated with cell-specific antigen and dispersed into the mixture to react with the cells of interest. The beads that bind to a cell effectively imbue that cell with a magnetic moment.
In practice, an operator pours the cell-containing fluid mixture through the MACS magnetic filter that collects the cells bound to the magnetic particles. The filter is formed as a titration column seated between the poles of a permanent magnet. Located within the column is a ferromagnetic steel wool mesh of select porosity. The magnet magnetizes the steel wool and as the fluid mixture flows through the steel wool, the cells bound to magnetic particles collect against the steel wool. Once the mixture has passed through the filter, the column is unseated from the magnet and the steel wool demagnetizes. The collected cells within the steel wool are eluted by a strong wash passing through the column and dislodging the cells from the steel wool mesh.
The cells collect in the wash fluid to form a target rich fluid suspension. In a subsequent step, the cells in the fluid are analyzed using flow cytometry and statistical data regarding the target cell population is collected.
Although magnetic filtering offers a general assay for magnetically collecting particles from a fluid mixture, it still suffers from the disadvantages generally associated with the filtering process. In particular, the filtering technique is a time-consuming and laborious process that typically requires multiple repetitions of the filtering step in order to sufficiently isolate the particles from the mixture. Therefore, the filtering technique is not a particularly efficient method for isolating rare particle sub-populations.
Furthermore, the filtering technique is fairly non-specific in that it collects the magnetic particles along with particles that are sized to be captured in the steel wool mesh. As such, the magnetic filtering technique is poorly suited for extracting a pure subset of the target cell population. Similarly, the filtering technique is fairly limited to fluid samples that are sufficiently fluid to flow through the steel wool mesh and is difficult to use with more viscous samples.
In addition to being non-specific, the magnetic filter technique is nonquantitative and provides limited control over the number of target particles collected from the fluid mixture. As such, the magnetic filtering technique is poorly suited for extracting a specific quantity of target particles.
Furthermore, the filtering technique relies on a washing procedure that removes the particles from the filter and collects the particles into a wash fluid. During this procedure the wash fluid is typically contaminated by the steel-wool filter mesh. Therefore, before analysis of the isolated particles can begin, a subsequent step is necessary to separate the particles out of the contaminated wash fluid. As such, the magnetic filtering technique fails to provide a one-step system that can isolate target particles from a mixture and yield particles in a form suitable for immediate laboratory analysis.
In view of the foregoing, an object of the present invention is to provide methods and systems for extracting a specific particle sub-population from a mixture, and more particularly, to provide improved methods and apparatus for magnetically extracting particles from a mixture.
A further object of the present invention is to provide a magnetic separation system that detects the collection of target particles against a collection surface and that is further capable of indicating the number of target particles being collected from a mixture.
Still another object of the present invention is to provide a mechanism for extracting target particles from a mixture in a manner that achieves a select spatial distribution suitable for transfer to an optical element.
Yet another object of the invention is to provide improved methods and apparatus for extracting cells from a mixture that is facile to use and has a reduced number of steps.
Yet a further object of the invention is to provide an improved method and apparatus for magnetically extracting a target cell population from a mixture in a manner that reduces the number of vessels contaminated by the mixture.
These and other objects of the invention are evident in the figures and description that follow.